Target (protein/gene name): serine/threonine protein phosphatase *NCBI Gene # or RefSeq#: GI:237839921 *Protein ID (NP or XP #) or Wolbachia#: XP_002369258.1 *Organism (including strain): Toxoplasma Gondii Etiologic Risk Group (see link below): Risk Group 2 (RG2) - Parasitic Agents *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Toxoplasma Gondii is a eukaryotic parasitic protozoan that causes toxoplasmosis. This organism is found worldwide and capable of infecting all warm-blooded animals. As one of the most common human parasites, T. Gondii has been predicted to have infected up to a third of the global population. Although the parasite is found worldwide, the disease itself (toxoplasmosis) only causes mild flu-like symptoms. However in infants and HIV/AIDS patients, this infection cause serious and potentially fatal illness. Infection of this disease usually occurs due to consumption of raw/undercooked meat, ingesting soil/vegetables/water contaminated with cat feces, or through transmission from mother to fetus during pregnancy if mother is infected. Essentiality of this protein: Essential http://www.sciencedirect.com/science/article/pii/S1286457909001403 http://ec.asm.org/content/6/1/73.full http://www.molbiolcell.org/content/14/5/1900.full.pdf+html Complex of proteins?: None Druggable Target: No known targets, 0.3 Druggability index http://www.tdrtargets.org/targets/view?gene_id=264187
Image of protein (PyMol with features delineated and shown separately):
Figure 1. Pymol image of 2I44 shown as lines and ribbons. The 3 different chains are labeled in pink, green, and cyan.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
1 magiegpvag egaggaalgs cegpgenatk aassmslldr qieqlrrcec iperevkalc
61 akakeilvee snvqrvdvpv ticgdihgqf ydllelfrvg nflpetsylf lgdfvdrgyf
121 svetflflla lkvrypdrit lirgnhesrq itqvygfyde clrkygsstv wryctevfdy
181 lalaaiiddr lfalhgglsp aissvdeirs idrkqevphe gpmcdllwsd pedmqgwgys
241 prgagylfga divkafchtn nieiiarahq lvmdgykwwf gkklvtvwsa pnycyrcgnv
301 atvmeldeql nyqfktfeaa pperrgipsk kpppdyfl *length of your protein in Amino Acids-338 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 38032.3g Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 57465, assuming all pairs of Cys residues form cystines
Ext. coefficient 56840, assuming all Cys residues are reduced TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 2. The diagram above shows that the protein is in the cytosal. Scores above 500 are transmembrane proteins.
*GC% Content for gene: 56.35% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: GI:237839921
*Protein ID (NP or XP #) or Wolbachia#: XP_002369258.1
*Organism (including strain): Toxoplasma Gondii
Etiologic Risk Group (see link below): Risk Group 2 (RG2) - Parasitic Agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up): Toxoplasma Gondii is a eukaryotic parasitic protozoan that causes toxoplasmosis. This organism is found worldwide and capable of infecting all warm-blooded animals. As one of the most common human parasites, T. Gondii has been predicted to have infected up to a third of the global population. Although the parasite is found worldwide, the disease itself (toxoplasmosis) only causes mild flu-like symptoms. However in infants and HIV/AIDS patients, this infection cause serious and potentially fatal illness. Infection of this disease usually occurs due to consumption of raw/undercooked meat, ingesting soil/vegetables/water contaminated with cat feces, or through transmission from mother to fetus during pregnancy if mother is infected.
Essentiality of this protein: Essential
http://www.sciencedirect.com/science/article/pii/S1286457909001403
http://ec.asm.org/content/6/1/73.full
http://www.molbiolcell.org/content/14/5/1900.full.pdf+html
Complex of proteins?: None
Druggable Target: No known targets, 0.3 Druggability index
http://www.tdrtargets.org/targets/view?gene_id=264187
*EC#: 3.1.3.16
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
No BRENDA information was found specifically to Toxoplasma Gondii.
http://www.sigmaaldrich.com/technical-documents/articles/biowire/phosphatase-inhibitor.html
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Kinase and Phosphatase Assay - Reagent:Oxadaic acid, EDTA, cyclosporin, FK-506.
http://www.molbiolcell.org/content/14/5/1900.full.pdf+html
-- List cost and quantity of substrate reagents and supplier
$68.30 - Okadaic Acid (from Sigma)
http://www.sigmaaldrich.com/catalog/product/sigma/o4511?lang=en®ion=US
$19.10- EDTA (from Sigma)
http://www.sigmaaldrich.com/catalog/product/sial/eds?lang=en®ion=US
Structure Available (PDB)
-- PDB #: 2I44
http://www.rcsb.org/pdb/explore/explore.do?structureId=2I44
Current Inhibitors: EDTA, Okadaic Acid, Cyclosporin, FK-506
Expression Information (has it been expressed in bacterial cells): E. Coli
http://ec.asm.org/content/6/1/73.full
Purification Method: Immunoaffinity purification
http://ec.asm.org/content/6/1/73.full
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
1 magiegpvag egaggaalgs cegpgenatk aassmslldr qieqlrrcec iperevkalc
61 akakeilvee snvqrvdvpv ticgdihgqf ydllelfrvg nflpetsylf lgdfvdrgyf
121 svetflflla lkvrypdrit lirgnhesrq itqvygfyde clrkygsstv wryctevfdy
181 lalaaiiddr lfalhgglsp aissvdeirs idrkqevphe gpmcdllwsd pedmqgwgys
241 prgagylfga divkafchtn nieiiarahq lvmdgykwwf gkklvtvwsa pnycyrcgnv
301 atvmeldeql nyqfktfeaa pperrgipsk kpppdyfl
*length of your protein in Amino Acids-338 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 38032.3g
Molar Extinction coefficient of your protein at 280 nm wavelength:
Ext. coefficient 57465, assuming all pairs of Cys residues form cystines
Ext. coefficient 56840, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
atggcgggcattgaaggcccggtggcgggcgaaggcgcgggcggcgcggcgctgggcagc tgcgaaggcccgggcgaaaacgcgaccaaagcggcgagcagcatgagcctgctggatcgc cagattgaacagctgcgccgctgcgaatgcattccggaacgcgaagtgaaagcgctgtgc gcgaaagcgaaagaaattctggtggaagaaagcaacgtgcagcgcgtggatgtgccggtg accatttgcggcgatattcatggccagttttatgatctgctggaactgtttcgcgtgggc aactttctgccggaaaccagctatctgtttctgggcgattttgtggatcgcggctatttt agcgtggaaacctttctgtttctgctggcgctgaaagtgcgctatccggatcgcattacc ctgattcgcggcaaccatgaaagccgccagattacccaggtgtatggcttttatgatgaa tgcctgcgcaaatatggcagcagcaccgtgtggcgctattgcaccgaagtgtttgattat ctggcgctggcggcgattattgatgatcgcctgtttgcgctgcatggcggcctgagcccg gcgattagcagcgtggatgaaattcgcagcattgatcgcaaacaggaagtgccgcatgaa ggcccgatgtgcgatctgctgtggagcgatccggaagatatgcagggctggggctatagc ccgcgcggcgcgggctatctgtttggcgcggatattgtgaaagcgttttgccataccaac aacattgaaattattgcgcgcgcgcatcagctggtgatggatggctataaatggtggttt ggcaaaaaactggtgaccgtgtggagcgcgccgaactattgctatcgctgcggcaacgtg gcgaccgtgatggaactggatgaacagctgaactatcagtttaaaacctttgaagcggcg ccgccggaacgccgcggcattccgagcaaaaaaccgccgccggattattttctg
*GC% Content for gene: 56.35%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):